SUMMARY

In teleosts, the thyroid gland is mostly found in the subpharyngeal region.
However, in several species thyroid follicles are found in, for example,
heart, head kidney and kidney. Such heterotopic thyroid follicles are active,
and considered to work in concert with the subpharyngeal thyroid. In
Mozambique tilapia (Oreochromis mossambicus) thyroid activity is,
indeed, restricted to the subpharyngeal region; in common carp (Cyprinus
carpio) the functional endocrine thyroid is associated with renal
tissues. The subpharyngeal follicles of carp comprise only 10% of the total
thyroid tissue, and these follicles neither accumulate iodide nor synthesize
or secrete thyroid hormones to a significant degree. Although the shape and
size of carp subpharyngeal and renal follicles vary, the epithelial cell
height of the thyrocytes and thyroxine immunoreactivity do not differ, which
suggests that the activity of the carp subpharyngeal thyroid follicles is
dormant. Differences in thyroid physiology between the two fish species were
further assessed at the level of peripheral thyroid hormone metabolism. Carp
clears plasma of thyroid hormones faster than tilapia does. Furthermore, a
significant amount of conjugated thyroid hormones was observed in the plasma
of tilapia, which was preceded by the occurrence of thyroid hormone conjugates
in the subpharyngeal region and coincides with the appearance of conjugates in
the surrounding water. Apparently, plasma thyroid hormone conjugates in
tilapia originate from the thyroid gland and function in the excretion of
thyroid hormones. Our data illustrate the variability in teleostean
thyroidology, an important notion for those studying thyroid physiology.

Introduction

The main products of the thyroid gland are thyroid hormones, the actions of
which are pleiotropic and involve the regulation of metabolism, growth and
development, including metamorphosis. Thyroid hormones are synthesized in
thyroid follicles, the functional units of the thyroid gland, composed of
thyrocytes enclosing a protein-filled colloid matrix. Thyroid-stimulating
hormone (TSH) from the pituitary gland is the major stimulus for thyroid
hormone synthesis and release (Blanton and
Specker, 2007; Eales and
Brown, 1993). Plasma thyroid hormone levels are not only
determined by thyroid hormone synthesis and secretion but also by peripheral
metabolism (viz. deiodination and conjugation), clearance and
excretion of thyroid hormones. Thyroid hormones are generally excreted as
glucuronide or sulphate conjugates via the bile
(Finnson and Eales, 1996).
Unlike the compact mammalian thyroid gland, the thyroid gland of most
teleostean fish consists of non-encapsulated follicles scattered in the
subpharyngeal region surrounding the ventral aorta
(Gudernatsch, 1911). In
several species of fish, however, heterotopic thyroid follicles, i.e.
follicles located outside the typical subpharyngeal region, have been reported
(Baker, 1958).

Heterotopic thyroid follicles can be found near or in the heart, spleen,
liver, oesophagus, brain and choroid rete mirabile of fish, but are generally
restricted to tissues that ontogenetically derive from renal primordia,
viz. the head kidney (pronephros) and the adult kidney
(opistonephros) (Baker, 1958).
Thyroid heterotopia has been described in species throughout the Teleostei
infraclass; it is found in representatives of the order of anchovies and
herrings (Clupeiformes, 1 species), catfish (Siluriformes, 4 species),
killifish (Cyprinodontiformes, 3 species), swamp eels (Synbranchiformes, 1
species), perch-like fish (Perciformes, 3 species), rainbow fish and
silversides (Atheriniformes, 1 species), and minnows and suckers
(Cypriniformes, 14 species). Interestingly, 13 of the 27 fish species in which
heterotopic thyroid follicles have been described belong to the family of carp
and minnows (Cyprinidae), including species such as goldfish (Carassius
auratus) and common carp.

Since iodide is exclusively incorporated into thyroid hormones and its
metabolites, the use of radioactive isotopes of iodide offers unique
possibilities for the investigation of thyroid hormone dynamics.
Autoradiography of the thyroid gland in Mozambique tilapia (Oreochromis
mossambicus Peters) and common carp (Cyprinus carpio L.)
serendipitously revealed differences in iodide metabolism, and pointed to the
presence, in carp, of heterotopic thyroid tissue that is functionally
different from that in the subpharyngeal region. This was the motivation for
the studies described here.

Materials and methods

Animals

Common carp (Cyprinus carpio L.) of the all-male E4×R3R8
isogenic strain (Bongers et al.,
1998) were obtained from the Department of Fish Culture and
Fisheries of Wageningen University, The Netherlands. Mozambique tilapia
(Oreochromis mossambicus Peters) were obtained from laboratory stock.
Fish were kept in 150 l tanks with aerated, recirculating city of Nijmegen tap
water, at a photoperiod of 16 h light and 8 h darkness at 23°C for carp
and 27°C for tilapia. Fish were fed commercial fish food (Trouvit, Trouw,
Putten, The Netherlands) at a daily ration of 1.5% of their estimated body
weight. Animal handling followed approved university guidelines.

Whole-body autoradiography

Juvenile carp and tilapia (standard length 6–8 cm) were exposed for
16.5 h to 250 μCi Na125I, which was added to the aerated water
in a 3 l tank, at 23 and 27°C, respectively. Thyrostatic potassium
perchlorate (KClO4) was added at a concentration of 1 mmol
l–1, and its effect on 125I uptake was compared
with that in an untreated group. After exposure, fish were deeply
anaesthetized with 0.1% (v/v) 2-phenoxyethanol and killed by immersion in
melting isoflurane (–70°C). Animals were embedded in 5%
carboxymethyl cellulose and stored at –20°C, and whole-body
crysosections were obtained according to a method described by Rijntjes et al.
(Rijntjes et al., 1979). In
short, a carboxymethyl cellulose block containing a specimen was mounted on
the stage of a LKB 2250-PMV cryomicrotome (LKB, Stockholm, Sweden). Sections
were collected with the aid of cellulose tape that was applied to the upper
surface of the carboxymethyl cellulose block, and were freeze dried in the
microtome for 24 h. Sections 30 μm thick were taken every 90 μm.
Freeze-dried whole-body sections of the whole fish were placed on Biomax MR-1
X-ray film (Eastman Kodak Company, Rochester, NY, USA); films were exposed for
3 days at –70°C after which they were developed according to the
manufacturer's protocol.

Injection procedure and sampling

Carp (102±14 g; N=24) and tilapia (117±17 g;
N=24) were injected intraperitoneally (i.p.) with 20.3 μCi
carrier-free Na125I (Amersham Biosciences, Amersham, Bucks, UK) per
100 g body weight. The 125I specific activity was
82×1015 Bq mol–1 and the radiotracer was
dissolved in 0.9% NaCl. After injection, fish were immediately transferred to
individual tanks with 3.5 l aerated city of Nijmegen tap water. During the
experiment, water was sampled and radioactivity was measured. Fish were
sampled at set times after injection by adding 0.1% (v/v) 2-phenoxyethanol to
the individual tanks to induce anaesthesia. Blood was sampled by puncture of
the caudal vessels with a heparinized syringe fitted with a 23 G needle and
plasma was collected after centrifugation at 4°C (4000 g,
15 min). The fish were then killed by spinal transection and selected organs
and tissues, as indicated in the figure legends, were collected. All tissues
and the remaining carcass were weighed and the volume and weight of total bile
and the collected plasma sample were determined. The radioactivities of bile
and plasma were measured in an LKB 1272 Clinigamma γ-counter (Wallac,
Turku, Finland) and immediately thereafter subjected to Sephadex LH-20
chromatography (see below). All tissues were homogenized according to Chopra
et al. (Chopra et al., 1982)
with an all-glass Potter-Elvehjem homogenization device in ice-cold 0.1 mol
l–1 Tris-HCl buffer (pH 8.7), added at 6 ml
g–1 tissue. Total radioactivity of the homogenates was
determined as described for bile and plasma. The carcass was microwaved for 3
min at 800 W and homogenized in a blender in 200 ml 0.1 mol
l–1 Tris-HCl buffer (pH 8.7). The resulting total volume was
assessed and the radioactivity of sextuplicate 1 ml subsamples was
determined.

Histochemistry

The subpharyngeal region, head kidney and kidney of four carp and tilapia
(39.3±0.5 g) were fixed in Bouin's solution for 24 h. Tissues were
dehydrated in a graded series of ethanol, embedded in paraplast and sectioned
at 7 μm. Every 140 μm, two serial sections were collected and mounted on
glass slides. Sections were stained with a modified Crossmon's connective
tissue stain (Crossmon, 1937)
as follows: 1.3 g l–1 Light Green SF yellowish
(Chroma-Gesellschaft, Stuttgart, Germany), 1.7 g l–1 Orange G
(Searle Diagnostic, High Wycombe, Bucks, UK) and 2 g l–1 acid
fuchsin (Fuchsin S from Chroma-Gesellschaft) were dissolved in distilled water
at 80°C. The solution was cooled to room temperature, and 1 g of
phosphotungstic acid hydrate was added to a 50 ml volume, followed by 2 ml
glacial ethanoic acid and 100 ml absolute ethanol. The final solution was
filtered and stored. Crossmon's trichrome stain was followed by a haematoxylin
counterstain. Using this procedure, the colloid in thyroid follicles stains
bright orange-red, which facilitates digital analysis of images.

Morphological data analysis

Sections were analysed with a Leica DM-RBE light microscope (Leica,
Wetzlar, Germany). Each thyroid follicle in the section was digitally
photographed at 20-times magnification. The colloid in every follicle was
manually selected using Adobe Photoshop 7.0 software and quantified using
MetaMorph 6 software (Universal Imaging, Downingtown, PA, USA). The epithelial
cell height of three thyrocytes per follicle in five follicles per tissue per
fish was digitally determined. The area, perimeter, maximal diameter, length
and width of every single colloid cross-section were measured. The shape of
the colloid was described with three dimensionless shape descriptors: form
factor, roundness and aspect ratio, which were calculated as follows
(Ponton, 2006):
where Area and Perimeter are the measured area (μm2) and
perimeter (μm) of a colloid, respectively. The form factor expresses the
evenness of the colloid's outline; as its value approaches 1, so the outline
resembles more the outline of a circle.
where Area and Maximal diameter are the measured area (μm2) and
maximal diameter (μm), respectively, of a colloid. A colloid with a maximum
roundness value of 1 perfectly resembles a circle.
The larger the aspect ratio, the more elongated the colloid is; a ratio of 1
corresponds to a perfectly circular colloid.

An initial analysis of frequency distributions revealed that form
descriptors were not Gaussian distributed, and we therefore chose the mode as
a descriptive statistic. To avoid subjective selection of bin width and
endpoint, we determined the frequency distribution by kernel density
estimation (Parzen, 1962)
using an add-in utility (version 1.0e) for Microsoft® Excel from the Royal
Society of Chemistry (Thompson,
2006) (see Fig. S1 in supplementary material).

In vitro incubations

Subpharyngeal tissue between the second and fourth gill arches, head kidney
and kidney was dissected from 14 carp (61±15 g). Tissues were weighed
and diced into approximately 1 mm3 fragments and immediately
transferred to a microtitre plate in 3 ml Hepes-Tris-buffered medium (pH 7.4)
saturated with carbogen (95% O2–5% CO2) and
allowed to recover for 1 h. Then, tissues were transferred to a clean plate in
3 ml of the aforementioned buffer and exposed to 10 mIU ml–1
bovine TSH (bTSH; Sigma) or saline vehicle. Tissues were incubated for 24 h at
23°C, after which the incubation medium was sampled. Total T4 (thyroxine,
or 3,5,3′5′-tetraiodothyronine) in the medium was determined using
a commercially available enzyme-linked immunoassay (Research Diagnostics,
Inc., Flanders, NJ, USA) according to the manufacturer's instructions.
Thyroxine-spiked samples gave representative readouts.

Thyroid hormone extraction

Several different methods based on extraction with Tris-HCl buffer,
ethanol, methanol, butanol or chloroform were tested. We found a combination
of Tris-HCl buffer and chloroform to be the most efficient in extracting
radioactivity. Homogenates from the subpharyngeal region, head kidney and
kidney tissues were obtained from intraperitoneally 125I-injected
animals, as described above. Then, 25 mg of pancreatin (Merck, Darmstadt,
Germany), suspended in 0.1 mol l–1 Tris-HCl buffer (pH 8.7),
was added to 1 ml of tissue homogenate as described by Tong and Chaikoff
(Tong and Chaikoff, 1957),
which was then incubated for 17 h at 35°C. Chloroform (1.5 ml) was added
and the incubate was vigorously mixed for 2 min and centrifuged at 4°C
(4000 g, 15 min). The water phase was collected by aspiration
and stored at –20°C until further analysis; 1 ml of 0.1 mol
l–1 Tris-HCl buffer (pH 8.7) was added to the remainder of
the chloroform–pancreatin mixture, and it was mixed for 10 min, and
incubated for 48 h at 4°C. The mixture was then centrifuged at 4°C
(4000 g, 15 min) and the water phase was aspirated and stored
at –20°C until further analysis. The extraction procedure was
repeated once, after which the radioactivity of the three pooled water phases
and of the remaining chloroform–pancreatin mixture was determined.

Sephadex LH-20 column chromatography

Sephadex LH-20 column chromatography was performed as described by Mol and
Visser (Mol and Visser, 1985).
In short, glass pipettes were filled with 1 ml Sephadex LH-20 (Amersham
Biosciences, Uppsala, Sweden) suspension in water (10% w/v) and equilibrated
with 3 × 1 ml volumes of 0.1 mol l–1 HCl. Samples (100μ
l) of plasma, bile and extracts of the subpharyngeal region, head kidney
and kidney were deproteinized with 4 volumes of methanol and centrifuged at
4°C (4000 g, 15 min). The supernatants were acidified with
1 volume of 1 mol l–1 HCl and loaded on to the column. The
samples were then eluted from the column with 2× 1 ml volumes of 0.1 mol
l–1 HCl to separate free iodide, 6× 1 ml volumes of
H2O to separate water-soluble conjugated forms of iodothyronines,
and 3× 1 ml volumes of 1 mol l–1 NH3/ethanol
to separate native iodothyronines. The radioactivity of the collected
fractions was measured in a γ-counter.

Results

Autoradiography

Autoradiography demonstrated 125I in the subpharyngeal region of
tilapia. The radioactivity we observed in the intestinal tract was most
probably caused by drinking (Fig.
1A,B). The inhibitory effect of perchlorate
(Fig. 1H) hints at the
involvement of the sodium–iodide symporter in the accumulation of
radioiodide in tilapia intestine. In carp, 125I was evident in the
kidney but not in the subpharyngeal region
(Fig. 1C,D). Furthermore, the
gall bladder of carp contained radioactivity
(Fig. 1E,F), which is in
contrast with tilapia. Exposure to perchlorate blocked iodide accumulation in
the subpharyngeal region of tilapia (Fig.
1G,H) and carp kidney, although radioactivity was still present in
carp gall bladder (Fig.
1I,J).

Representative autoradiographs of 30 μm, whole-body cryosections of
juvenile tilapia (A,B) and juvenile carp (C–F) exposed to
125I, and of tilapia (G,H) and carp (I,J) exposed to
125I and KClO4. Broken circles indicate the position of
the subpharyngeal region (1), the gall bladder (2) and the kidney (3). Scale
bars, 1 cm.

125Iodide tissue distribution

We retrieved 97±14% of the nominal amount of injected radioactivity
from the tissue extracts. Measured 2 h after injection, the plasma
125I radioactivity in tilapia was
719(±290)×103 c.p.m. g–1, which
decreased to 151(±110)×103 c.p.m. g–1
at 96 h. In carp, these values were 1190(±230)×103
c.p.m. g–1 and 22(±12)×103 c.p.m.
g–1, respectively. The subpharyngeal region in tilapia
maximally accumulated 125I 31-fold over the plasma level at 96 h
into the chase (Table 1). All
tissues other than the subpharyngeal region showed a decrease in radioactivity
during this period (Fig. 2A).
In carp, kidney, head kidney and bile were the only compartments where
radioactivity accumulated (Fig.
2B). In carp, kidney tissue was able to accumulate 125I
more than 500-fold relative to plasma radioactivity at 96 h. The head kidney
and subpharyngeal region of carp also accumulated iodide, 91- and 20-fold,
respectively, compared with plasma (Table
1). While carp bile showed a 37-fold accumulation of
125I at 96 h, tilapia bile radioactivity increased only twofold
(Table 1).

Tissue distribution of the radioactivity, shown as the percentage of the
total dose injected in the fish per gram of tissue at 2, 24, 48 and 96 h after
i.p. 125I injection in tilapia (A, N=6) and carp (B,
N=6). Results are means ± s.d. One-way ANOVA was used for
statistical evaluation. Asterisks show significant difference compared with
the radioactivity level at 2 h (*P<0.05,
**P<0.01, ***P<0.001).

Histology and morphological analysis

We could only detect thyroid follicles in the subpharyngeal region of
tilapia, and this observation is corroborated by the 125I tissue
distribution shown in Fig. 2A.
In carp, thyroid follicles were observed in kidney, subpharyngeal region and
head kidney (Fig. 3A–C).
The bright red stain of the lumen of follicular structures obtained with
Crossmon's trichrome colocalized with a specific thyroxine immunoreactivity,
confirming that the follicles found in all three tissues were indeed thyroid
follicles (Fig. 3D–F). On
average, 1391±196 (N=4) follicle cross-sections were observed
in carp, of which 87±2% was located within the kidney, 3±1%
within the head kidney and 10±2% within the subpharyngeal region.

The modal value of the area of the kidney colloid was significantly smaller
than that of the colloid in the subpharyngeal region and head kidney
(Table 2). Also, the mode of
the perimeter of the colloid was significantly smaller in the kidney follicles
as compared with head kidney follicles
(Table 2). Although the mode of
the form factor of the colloid did not differ between the tissues, the mode of
the roundness and aspect ratio did; the colloid in the subpharyngeal region
was significantly rounder and less elongated than the colloid in the head
kidney (Table 2). No
differences (P=0.192) were observed between the epithelial cell
height of the subpharyngeal region (6.04±0.12 μm, N=4),
head kidney (6.67±0.68 μm, N=4) and kidney follicles
(6.54±0.43 μm, N=4).

TSH-mediated T4 release in vitro

Exposure to 10 mIU ml–1 bTSH for 24 h significantly
stimulated the release of T4 from carp kidney and head kidney tissue, 1.7- and
3.6-fold, respectively (Fig.
4). Tissue from the subpharyngeal region was unresponsive to 10
mIU ml–1 bTSH.

125Iodide pulse chase

Radiolabelled compounds were extracted with very high recoveries from the
subpharyngeal region (92±2% of total radioactivity), head kidney
(77±11%) and kidney tissue (92±5%) in tilapia. Similar
efficiencies were obtained for carp tissues (99±1%, 96±2% and
96±2%, respectively). In previous studies, extraction of radiolabelled
thyroid hormones from tissues with ethanol or methanol combined with
chloroform usually yielded recoveries of less than 70%, and ranging from 45%
to 94% (Crane et al., 2004;
Krysin, 1990;
Szisch et al., 2005;
Tagawa and Hirano, 1987).

Chromatographic analysis of plasma revealed that the decrease in total
radioactivity, as observed in Fig.
2, can mainly be attributed to a decrease of the tracer injected
(125I), which occurs at a faster rate in carp than in tilapia;
between 2 and 48 h after injection, the 125I plasma level had
decreased by 90±6% in carp, whereas in tilapia, it had decreased by
48±31% (Fig. 5).
Radiolabelled, i.e. de novo synthesized, thyroid hormones appeared in
increasing amounts in tilapia plasma during the experimental chase. In carp,
however, newly synthesized thyroid hormones decreased from 2 h onwards.
Conjugated forms of thyroid hormones appeared in tilapia plasma following the
appearance of newly synthesized thyroid hormones.

The chronology of the appearance of labelled thyroid hormone metabolites in
the subpharyngeal region, the head kidney and the kidney differed markedly
between tilapia and carp. After an initial accumulation, the iodide level
remained essentially constant as of 24 h in the subpharyngeal tissue of
tilapia, whereas levels of labelled thyroid hormones and conjugates increased
after 24 h (Fig. 6A). In kidney
(Fig. 6B) and head kidney
tissue (Fig. 6C) of tilapia no
accumulation of iodide was observed. Small amounts of labelled thyroid
hormones and conjugates appeared in these tissues at 48 h, which corresponds
with the chronology seen in plasma, suggesting that these compounds originate
from plasma.

In carp, maximum radioiodide accumulation in the kidney was reached at 48
h, after which the iodide level remained stable at 96 h
(Fig. 6B). Thyroid hormones and
thyroid hormone conjugates increased from 24 h onwards, reaching maximum
levels at 48 h. Also in carp head kidney, newly synthesized thyroid hormones
were observed as of 24 h, while thyroid hormone conjugates were essentially
absent in this tissue (Fig.
6C). The subpharyngeal region of carp did not accumulate
detectable radioiodide and virtually no radiolabelled thyroid hormones and
thyroid hormone conjugates were observed
(Fig. 6A).

Radioactivity of iodide, thyroid hormone and thyroid hormone conjugates
fractions, shown as the percentage of the total dose remaining in the fish at
the time of sampling per gram of plasma, after Sephadex LH-20 chromatography
at 2, 24, 48 and 96 h after i.p. 125I injection in tilapia (•,
N=6) and carp (○, N=6). Results are means ± s.d.
One-way ANOVA was used for statistical evaluation. Significantly different
levels of radioactivity compared with that at 2 h are indicated for tilapia
(*P<0.05, **P<0.01,
***P<0.001) and carp (+P<0.05,
++P<0.01, +++P<0.001).

Chromatographic analysis of bile revealed an increase in radioiodide
content in carp bile, whereas in tilapia bile, the radioiodide level did not
change significantly (Fig. 7A).
Thyroid hormones and conjugated forms of thyroid hormone accumulated in the
bile of both species. The average total volume of the bile of both fish
species did not decrease during the experiment, indicating that the gall
bladder had not emptied in the intestinal tract during the chase period of the
experiment.

In the ambient water of both fish, iodide, thyroid hormones and thyroid
hormone conjugates were observed (Fig.
7B). Equal amounts of iodide and thyroid hormones were excreted by
the two fish species. Conjugated thyroid hormones, however, were only excreted
by tilapia.

Discussion

We observed remarkable differences in the location of active endocrine
thyroid tissue between carp and tilapia. In carp, three thyroid follicle
populations, active and inactive, were identified in the subpharyngeal region,
head kidney and kidney. Also, differences at the level of the peripheral
metabolism of thyroid hormones were observed. Not only did plasma clearance
differ but also the route of thyroid hormone excretion varied between carp and
tilapia.

Radioactivity of iodide, thyroid hormone and thyroid hormone conjugates
fractions, shown as the percentage of the total dose remaining in the fish at
the time of sampling per gram of tissue, in the subpharyngeal region (A),
kidney (B) and head kidney (C), after Sephadex LH-20 chromatography at 2, 24,
48 and 96 h after i.p. 125I injection in tilapia (•,
N=6) and carp (○, N=6). Results are means ± s.d.
One-way ANOVA was used for statistical evaluation. Significantly different
levels of radioactivity compared with that at 2 h are indicated for tilapia
(*P<0.05, **P<0.01,
***P<0.001) and carp (+P<0.05,
++P<0.01, +++P<0.001).

In tilapia, the subpharyngeal region was the only site in which thyroid
follicles were found, where perchlorate-sensitive iodide accumulation was
observed, and where thyroid hormones were synthesized de novo. This
demonstrates, for tilapia, a location and activity typical for the teleostean
thyroid gland. The anatomical location of the thyroid gland in carp, however,
deviates from that in tilapia. In carp, the renal tissues display thyroid
activity as evidenced by iodide accumulation, confirming the observations of
Leray and Febvre (Leray and Febvre,
1968) and Lysak (Lysak,
1964). Although both head kidney and kidney in carp synthesized
thyroid hormones and secreted thyroid hormones following TSH stimulation, the
head kidney can have only a moderate share in total thyroid output. Not only
was the kidney the foremost iodide-accumulating tissue in carp, which was
inhibited by perchlorate, it also harbours the largest amount of thyroid
tissue: 87% of the total thyroid follicle population, as opposed to 3% in the
head kidney. This suggests a significant role for the kidney thyroid follicles
in thyroid economy of carp.

The most striking aspect of this study is the absence of iodide
accumulation and thyroid hormone synthesis in the subpharyngeal region of
carp, despite the presence of thyroid follicles. Furthermore, we found that
carp subpharyngeal thyroid follicles do not have the capacity to release
thyroid hormones upon stimulation with TSH in vitro, whereas renal
thyroid follicles do. This, together with the high prevalence of thyroid
follicles, establishes the kidney as the anatomical site of the thyroid gland
in this species. These results may pose questions as to whether to
(re-)consider the term `heterotopic' in conjunction with `thyroid follicles'
in common carp. In goldfish (Carassius auratus), a species closely
related to common carp, the subpharyngeal thyroid follicles are active and are
responsible for 11–40% of total iodide accumulation
(Chavin and Bouwman, 1965;
Peter, 1970), leaving a
considerable role for subpharyngeal thyroid follicles in the uptake of iodide
in this species. However, the subpharyngeal follicles in goldfish appear not
to be responsive to T4 treatment; changes in thyroid activity, i.e. iodide
accumulation and epithelial cell height, are primarily mediated through
inter-renal thyroid follicles, which shows that the thyroid populations are
not physiologically equivalent (Peter,
1970).

Radioactivity of iodide, thyroid hormone and thyroid hormone conjugates
fractions, shown as a percentage of the total dose remaining in the fish at
the time of sampling per gram of tissue, in the bile (A) and the ambient water
(B), following Sephadex LH-20 chromatography at 2, 24, 48 and 96 h after i.p.
125I injection in tilapia (•, N=6) and carp (○,
N=6). Results are means ± s.d. One-way ANOVA was used for
statistical evaluation. Significantly different levels of radioactivity
compared with that at 2 h are indicated for tilapia
(**P<0.01, ***P<0.001) and carp
(+P<0.05, ++P<0.01, +++P<0.001).

Histologically, the subpharyngeal follicles in carp appear normal, active
and similar to kidney thyroid follicles, as evidenced by the epithelial cell
height, which does not differ significantly from that of kidney follicles.
Differences in size and shape were observed between the thyroid follicle
populations, viz. the colloid of kidney follicles is the smallest,
and subpharyngeal follicles appear to be more round than renal follicles. It
is tempting to interpret the small colloidal area of renal thyroid follicles
as an indication of increased colloidal resorption, which, again, is
indicative of increased hormonogenesis in these follicles. The latter is
corroborated by our observation of increased iodide accumulation and the
presence of de novo synthesized thyroid hormones in this region.

Studies from Raine and colleagues
(Raine and Leatherland, 2000;
Raine et al., 2005) show that
in embryonic rainbow trout (Oncorhynchus mykiss) the functional unit
of the thyroid gland appears tubular, not follicular, and that this morphology
is retained in the juvenile stages. Despite the fact that carp thyroid tissues
were sectioned in varying planes, we did not detect tubular structures in our
serial sections. A significant proportion of tubular colloid-filled structures
would also have been made apparent by a larger variation in the morphology
descriptors of the thyroidal colloid. Instead, they show little variation. Of
course, it remains to be determined whether measurement of thyroidal colloid
accurately reflects thyroid follicle morphology. Still, one can speculate that
differences in morphometrics might reflect differences in organogenesis
between species.

Although the subpharyngeal thyroid follicles in carp did not incorporate
radioiodide to a significant degree or synthesize thyroid hormones de
novo, these subpharyngeal follicles do show T4 immunoreactivity.
Apparently these follicles do have an intrinsic capacity to synthesize thyroid
hormones. Whether the rate of thyroid hormone synthesis is too slow to detect
within the 96 h of the experimental chase, or whether these follicles were
active during earlier life stages and are now dormant (and still contain T4)
remains to be determined. Interestingly, neotenic urodeles are able to
complete a full life cycle without metamorphosis
(Rosenkilde and Ussing, 1996).
Because of an impaired thyroid system these amphibians are unable to release a
surge of thyroxine, necessary to initiate metamorphosis. Although intact and
functional, their thyroid system is relatively inactive at several levels of
the thyroid axis, from the central regulation of the thyroid gland to the
peripheral deiodination of thyroid hormones. Neoteny has also been described
in fish; during adult life the ice goby (Leucopsarion petersii)
exhibits several larval characteristics, indicative of an impaired
metamorphosis. During its development, the thyroid follicles are smaller and
have a lower epithelial cell height when compared with a metamorphic goby
species; also no TSH immunoreactivity was observed in the pituitary
(Harada et al., 2003).
Although carp are not neotenic, further research on the carp subpharyngeal
thyroid follicles may provide more insight into the mechanisms controlling the
non-pharmacologically induced inactivity of the thyroid gland as observed in
neotenic organisms. A possible mechanism could be the temporal expression of
active and/or inactive splice variants of key regulators of thyroid hormone
synthesis, e.g. TSH receptor, sodium–iodide symporter or
thyroglobulin.

It is unclear why the functional endocrine thyroid tissue is located in the
kidney and not in the subpharyngeal region. We hypothesize that two,
potentially functional, thyroid populations with different sensitivities to
thyrotropic factors, or with different synthesizing properties, confer an
accurate regulation of thyroid gland output in response to a demand for
systemic thyroid hormone. It can also be hypothesized, regarding the close
juxtaposition of the extra-pharyngeal thyroid follicles to specific cell types
in the head kidney and kidney, that thyroid hormones have a paracrine effect
on inter-renal (cortisol-producing), chromaffin (catecholamine-producing),
and/or haematopoietic cells or on nephron structures. Paracrine relationships
between the stress axis and immune system have already been demonstrated in
the multifunctional carp head kidney (Metz
et al., 2006). Attempts to demonstrate a direct in vitro
effect of thyroid hormones on the release of cortisol in carp head kidney have
not been successful yet, even though treatment of carp with thyroxine resulted
in a decrease in the level of plasma cortisol
(Geven et al., 2006).

The presence of 125I radioactivity in carp bile and its absence
in tilapia bile suggests a faster turnover rate of thyroid hormones in carp
than in tilapia. This is corroborated by the faster clearance of iodide and
thyroid hormones from plasma, and the accumulation of iodide and thyroid
hormone conjugates in bile of carp compared with tilapia. Not considering
differences between species, these results appear to contradict the general
idea that higher temperatures result in increased thyroid activity
(Eales et al., 1982), as our
carp were held at a temperature that was 5°C lower than that for
tilapia.

The appearance of thyroid hormone conjugates in the plasma of tilapia is
consistent with the observations of DiStefano et al.
(DiStefano et al., 1998), who
found a significant amount of T3 glucuronides in plasma of Mozambique tilapia
after i.p. injection with [125I]T3
([125I]3,5,3′-triiodothyronine). Although thyroid hormone
sulphates are found in the sera of several mammals
(Santini et al., 1993;
Wu et al., 1992;
Wu et al., 1993), and indirect
evidence exists for thyroid hormone conjugates in the plasma of European
plaice (Pleuronectes platessa L.)
(Osborn and Simpson, 1969),
the Mozambique tilapia appears to be the only vertebrate in which plasma
thyroid hormone glucuronides are observed. By injection of trace amounts of
radioiodide instead of radiolabelled thyroid hormones, we circumvented the
possibility of altering the thyroid status of the fish. The injection of
radioiodide, as opposed to radiolabelled thyroid hormones, also allowed us to
speculate on the anatomical site at which conjugated thyroid hormones are
produced. Whereas thyroid hormone conjugates were observed in tilapia plasma
at 48 and 96 h after i.p. injection, they were already present in the
subpharyngeal region at 24 h, suggesting that the glandular thyroid itself may
be responsible for the production of thyroid hormone conjugates in tilapia
plasma. In this respect, the finding of conjugated forms of thyroid hormones
in the kidney of common carp, which harbours the functional thyroid, is
remarkable. However, we cannot exclude the possibility that cell types other
than thyrocytes, or tissues other than thyroid tissue, are responsible for the
presence of thyroid hormone conjugates.

The thyroid hormone conjugates in tilapia plasma have been suggested to
function as a pool of thyroid hormones from which, by deconjugation, a rapid
mobilization of bioactive thyroid hormones is available
(DiStefano et al., 1998). Our
results, however, suggest that the thyroid hormone conjugates in tilapia
plasma are involved in the excretion of thyroid hormones, through routes other
than bile. The appearance of thyroid hormone conjugates in the ambient water
coincides with the appearance of thyroid hormone conjugates in the plasma,
suggesting that thyroid hormone conjugates are excreted via plasma, possibly
through the gill or kidney. Since the volume of the gall bladders did not
decrease during the experiment and leakage of thyroid hormone conjugates over
the gall bladder wall in fish is negligible
(Collicutt and Eales, 1974),
the thyroid hormone conjugates in the ambient water are unlikely to originate
from bile. It is striking that in channel catfish (Ictalurus
punctatus), 8.1% of all injected [125I]T4 is excreted via
routes other than the gall bladder
(Collicutt and Eales, 1974)
and that in rainbow trout (Oncorhynchus mykiss), 8.2% and 6.7% of
injected [125I]T4 and [125I]T3, respectively, was
excreted via urine (Parry et al.,
1994). These reported percentages are of the same order of
magnitude as the observed percentage (8.3%) of thyroid hormone conjugates
found in tilapia plasma, supporting the idea that plasma thyroid hormone
conjugates in tilapia may function in the excretion of thyroid hormone
metabolites (DiStefano et al.,
1998).

In summary, we have shown that thyroid hormone synthesis, anatomical
location and activity of thyroid tissue, and thyroid hormone excretion in
teleost fish differ greatly between two species. The most distinct feature of
teleost thyroid physiology observed in this study is the presence of a
completely functional endocrine thyroid gland in the renal tissues of common
carp. This finding may open new possibilities for in vitro studies on
fish thyroid.

ACKNOWLEDGEMENTS

We thank Dr Liesbeth Pierson for technical assistance with morphometric
analyses, and Mr Nico Rijntjes for technical assistance with
cryosectioning.

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